Xylose is a most abundant carbohydrate existing in plant biomass and lumber and constitutes approximately 40% of lignocellulose substances. Xylose, in cellulose production steps, is formed as waste products from hydrolysates of xylan, a main component of hemicellulose. It is desired for effective use of carbon resources that xylose should be converted to ethanol or biomass by fermentation. Particularly, ethanol is being used in large amounts as liquid fuel.
Candida (Gong, C. S., Chen, L. F., Flickinger, M. C. and Tsao, G. T., Conversion of hemicellulose carbohydrate. Adv. Biochem. Eng. 20: 93-118 (1981); and Jeffries, T. W., Utilization of xylose by bacteria, yeast and fungi. Adv. Biochem. Biotech. 27: 1-32 (1983)), Debaryomyces, Hansenula, Kluyveromyces, Metschnikowia, Pachysolen, Paecilomyces (Wu, J. F., Lastick, S. M., Updegraff, D. M., Ethanol production from sugars derived from plant biomass by a novel fungus. Nature 321: 887-888 (1986)), Pichia (Maleszka, R. and Schneider, H., Fermentation of D-xylose, xylitol and D-xylulose by yeasts. Can. J. Microbiol. 28: 360-363 (1982)), and the like are known as yeasts that can utilize pentose (e.g., xylose or D-ribose) or the like.
In general, the conversion of pentose (e.g., xylose) to ethanol in organisms is mediated by pentose phosphorylation, and the resulted phosphorylated pentose is converted to ethanol through a pentose phosphate pathway. The pentose phosphorylation first requires the reduction of pentose accompanied by conversion from NADPH to NADP+, and this reaction is catalyzed by reductase. Pentitol, which is formed by the reduction of pentose, is subsequently subjected to oxidation accompanied by conversion from NAD+ to NADH. This reaction is catalyzed by dehydrogenase. D-pentulose is formed through the two-step reaction and phosphorylated by kinase to form pentose phosphate (Barnett, J. A., The utilization of sugars by yeasts. In: Advances in carbohydrate chemistry and biochemistry; ed., by Tipson, R. S. and Horton, D.; New York: Academic Press. 1976, pp. 125-235).
S. cerevisiae, a yeast predominantly used in bioethanol production, can utilize xylulose (pentulose) converted from xylose, while this yeast cannot ferment pentoses (Jeffries, T. W., Emerging technology for fermenting D-xylose. Trends in Biotechnology 3: 208-212 (1985)). What is important is that S. cerevisiae contains genes encoding pentose-fermenting proteins but does not express these genes.
The pentose fermentation by S. cerevisiae is thought to be possibly achieved by providing a xylose utilization pathway from a microorganism that metabolizes xylose. Many attempts have been made on S. cerevisiae to express bacterial xylose isomerase genes, and xylose fermentation, however, has ended unsuccessfully, presumably due to insufficient expression of the foreign genes (Sartny, A. V., McConaugh, B. L., Lodo, Z., Sundstrom, J. A., Furlong, C. E. and Hall, B. D., Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae. Appi. Eur. Microbiol. 53: 1996-2000 (1987); Amoer, R., Wilhelm, M. and Hollenberg, C. P., The fermentation of xylose—an analysis of the expression of Bacillus and Actinoplanes xylose isomerase genes in yeast. Appl. Microbiol. Biotechnol. 30: 351-357 (1989); Chan, E.-C., Ueng, P. P. and Chen, L. F., D-xylose fermentation to ethanol by Schizosaccharomyces pombe cloned with xylose isomerase gene. Biotech. Lett. 8: 231-234 (1986); and Chan, E.-C., Ueng, P. P. and Chen, L. F., Environmental effects on D-xylose fermentation by Schizosaccharomyces cerevisiae. Appl. Biotechnol. 20: 221-232 (1989)).
On the other hand, attempts as methods using yeast genes in another yeast have been made to isolate xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis which has the ability to utilize xylose, and to express the genes in S. cerevisiae (JP Patent Nos. 3122153 and 3193917). Moreover, attempts have been made on isolation of xylose reductase gene (Govinden, R., Pillay, B., van Zyl, W. H. and Pillay, D., Candida shehatae xylose reductase gene, complete cds., GenBank Direct Submission, Accession AF278715, (2000)) and purification of xylitol dehydrogenase (Yang, V. W. and Jeffries, T. W., Purification and properties of xylitol dehydrogenase from the xylose-fermenting yeast Candida shehatae. Appl. Biochem. Biotechnol., 26: 197-206 (1990)) from Candida shehatae, another yeast having the ability to utilize xylose. However, none of the cases have succeeded in isolating xylitol dehydrogenase genes from Candida shehatae. 